Rapid Detection of Group B Streptococcus andEscherichia coliin Amniotic Fluid Using Real-Time Fluorescent PCR

[摘要] Objective:To establish reliability and validity of real-time fluorescent PCR for early detection of bacterialinvasion of the amniotic cavity.Methods:Amniotic fluid samples from 40 patients undergoing mid-trimester genetic amniocentesis wereincubated for 6 h at 37℃ and were cultured on media specific for group B streptococcus (GBS) andE. coli.Concurrently, samples were analyzed with real-time fluorescent PCR (Roche LightCycler) using DNA primersand probes designed to detect the CAMP factor encodingcfbgene anduidAgene of GBS andE. coli,respectively. For positive control and to simulate amniotic fluid colonization, 104 cfu/ml of GBS andE. coliwereinoculated on sterile amniotic fluid and incubated for 6 h. Bacterial genomic DNA for the two organisms wasextracted and purified via the two-step precipitation method using a commercial kit. The real-time PCR assayswere also tested against 25 non-GBS and non-E. colibacterial species. The lower limit of detection for eachpathogen was established using serial dilution of bacterial genomic DNA.Results:All patient samples were negative for evidence of GBS andE. coliwith both culture and real-time PCRmethods. Amniotic fluid samples inoculated with GBS andE. coliwere positive with real-time PCR whereas the25 bacterial species other than GBS orE. colitested negative with the assay. Average total sample processingtime including the pre-enrichment step was 7 h 40 min. The average cost for DNA extraction and PCR testingwas $8.50 per test.Conclusion:Real-time fluorescent PCR is a valid and reliable method for detection of specific pathogens inamniotic fluid. This technique is sensitive for low inoculation levels. Real-time fluorescent PCR has potential toimpact clinical management as a rapid, reliable detection method for GBS andE. coliin chorioamnionitis.

[学科分类] 妇产科学